CD4⁺ Th1 cells play a critical role in the induction of cell-mediated immune responses that are important for the eradication of intracellular pathogens. Peptide-25 is the major Th1 epitope for Ag85B of Mycobacterium tuberculosis and is immunogenic in I-A^b mice. To elucidate the role of the TCR and IFN-γ/IL-12 signals in Th1 induction, we generated TCR transgenic mice (P25 TCR-Tg) expressing TCR α- and β-chains of Peptide-25-reactive cloned T cells and analyzed Th1 development of CD4⁺ T cells from P25 TCR-Tg. Naive CD4⁺ T cells from P25 TCR-Tg differentiate into both Th1 and Th2 cells upon stimulation with anti-CD3. Naive CD4⁺ T cells from P25 TCR-Tg preferentially develop Th1 cells upon Peptide-25 stimulation in the presence of I-A^b splenic antigen-presenting cells under neutral conditions. In contrast, a mutant of Peptide-25 can induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation is observed even in the presence of anti-IFN-γ and anti-IL-12. Furthermore, naive CD4⁺ T cells from STAT1 deficient P25 TCR-Tg also differentiate into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-A^b-transfected Chinese hamster ovary cells induce Th1 differentiation of naive CD4⁺ T cells from P25 TCR-Tg in the absence of IFN-γ or IL-12. These results imply that interaction between Peptide-25/I-A^b and TCR may primarily influence determination of the fate of naive CD4⁺ T cells in their differentiation towards the Th1 subset.